Anaerobically controlled expression system derived from the arcDABC operon of Pseudomonas aeruginosa: application to lipase production.

Details

Serval ID
serval:BIB_97F6541A08DD
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Anaerobically controlled expression system derived from the arcDABC operon of Pseudomonas aeruginosa: application to lipase production.
Journal
Applied and Environmental Microbiology
Author(s)
Winteler H.V., Schneidinger B., Jaeger K.E., Haas D.
ISSN
0099-2240 (Print)
ISSN-L
0099-2240
Publication state
Published
Issued date
1996
Volume
62
Number
9
Pages
3391-3398
Language
english
Abstract
The anaerobically inducible arcDABC operon encodes the enzymes of the arginine deiminase pathway in Pseudomonas aeruginosa. Upon induction, the arcAB mRNAs and proteins reach high intracellular levels, because of a strong anaerobically controlled promoter and mRNA processing in arcD, leading to stable downstream transcripts. We explored the usefulness of this system for the construction of expression vectors. The lacZ gene of Escherichia coli was expressed to the highest levels when fused close to the arc promoter. Insertion of lacZ further downstream into arcA or arcB did not stabilize the intrinsically unstable lacZ mRNA. On the contrary, lacZ mRNA appeared to be a vulnerable endonuclease target destabilizing arcAB mRNAs in the 5'-to-3' direction in P. aeruginosa. The native arc promoter was modified for optional expression in the -10 sequence and in the -40 region, which is a binding site for the anaerobic regulator ANR. In P. aeruginosa grown either anaerobically or with oxygen limitation in unshaken cultures, this promoter was stronger than the induced tac promoter. The P. aeruginosa lipAH genes, which encode extracellular lipase and lipase foldase, respectively, were fused directly to the modified arc promoter in an IncQ vector plasmid. Semianaerobic static cultures of P. aeruginosa PAO1 carrying this recombinant plasmid overproduced extracellular lipase 30-fold during stationary phase compared with the production by strain PAO1 without the plasmid. Severe oxygen limitation, in contrast, resulted in poor lipase productivity despite effective induction of the ANR-dependent promoter, suggesting that secretion of active lipase is blocked by the absence of oxygen. In conclusion, the modified arc promoter is useful for driving the expression of cloned genes in P. aeruginosa during oxygen-limited growth and stationary phase.
Keywords
Amino Acid Transport Systems, Anaerobiosis, Antiporters/genetics, Bacterial Outer Membrane Proteins/genetics, Bacterial Proteins/genetics, Base Sequence, Escherichia coli Proteins, Genes, Regulator, Genetic Vectors, Lac Operon, Lipase/biosynthesis, Molecular Sequence Data, Operon, Pseudomonas aeruginosa/genetics, Repressor Proteins
Pubmed
Web of science
Create date
25/01/2008 17:01
Last modification date
20/08/2019 14:59
Usage data